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Rapid Methods for the Extraction of Nucleic Acids from Biological Samples

Active Publication Date: 2016-11-17
LONGHORN VACCINES & DIAGNOSTICS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention introduces innovations that help quickly and easily collect and identify genes and nucleic acids of interest from a biological sample.

Problems solved by technology

This problem is magnified when a specimen is collected at a remote field site, or a significant distance from a doctor's office or laboratory environment, and especially where there may be limited or no access to refrigerator / freezer conditions.
Problems associated with the collection and handling of biological specimens are further exacerbated when the desired nucleic acids for downstream analysis include ribonucleic acid (RNA), which is particularly susceptible to degradation by endogenous or exogenous nuclease activity.
Another concern when working with biological specimens is the risk of release of infectious agents to individuals and the environment and, in addition, contamination to the biological specimen itself.
Inefficient recovery of nucleic acids requires repeated collection efforts, which are not always possible.

Method used

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  • Rapid Methods for the Extraction of Nucleic Acids from Biological Samples
  • Rapid Methods for the Extraction of Nucleic Acids from Biological Samples
  • Rapid Methods for the Extraction of Nucleic Acids from Biological Samples

Examples

Experimental program
Comparison scheme
Effect test

example 1

Formulation of Exemplary Storage Solutions

[0073]The present example provides a general formulation of the PSS compositions of the present invention. Additional formulations of the PSS compositions are exemplified in Examples 2 through 5.

Formulation Ranges of Exemplary Components for the Preparation of PrimeStore™ Compositions

[0074]

CompoundFinal Concentration Range (examples)A chaotrope, e.g.:Guanidine thiocyanateabout 0.5M to about 6Mand / or Guanidine hydrochlorideabout 0.5M to about 6Mand / or Guanidine isocyanateabout 0.5M to about 6MAn anionic detergent, e.g.:N-lauroyl sarcosine (inter alia Na salt)about 0.15% to about 1% (wt. / vol.)and / or Sodium dodecyl sulfate,about 0.15% to about 1% (wt. / vol.)Lithium dodecyl sulfate,about 0.15% to about 1% (wt. / vol.)Sodium glycocholate,about 0.15% to about 1% (wt. / vol.)Sodium deoxycholate,about 0.15% to about 1% (wt. / vol.)Sodium taurodeoxycholate, and / orabout 0.15% to about 1% (wt. / vol.)Sodium cholateabout 0.10% to about 1% (wt. / vol.)A reducing ag...

example 2

Formulation of an Exemplary Storage Solution

[0075]The present example describes a first exemplary formulation of the compositions of the invention. This formulation is alternatively referred to as “PrimeStore™ Solution” or “PSS” version 1.

Preparation of PrimeStore™ Composition (Version 1)

[0076]

CompoundFinal Conc.Guanidine thiocyanate4MSodium citrate30 mMSodium dodecyl sulfate0.25% (wt. / vol.)N-lauroyl sarcosine, sodium salt0.25% (wt. / vol.)2-mercaptoethanol (β-ME)0.1MAntifoam A0.1% (wt. / vol.)Citric acidq.s. to adjust pH to 6.5Nuclease-free water11.82 mLMagnetic NACM beads80 mg

example 3

Preparation of a Second Exemplary Storage Solution

[0077]The present example describes the preparation of another exemplary storage solution according to the present invention. This formulation may be referred to as PSS, which is PrimeStore™ version 2.

Preparation of PrimeStore™ Composition (Version 2)

[0078]

CompoundQuantityFinal Conc.Guanidine thiocyanate35.488 gm3MTCEP0.02867 gm1 mMSodium citrate0.2931 gm10 mMN-lauroyl sarcosine,0.5 gm0.5%sodium salt (NLS)Antifoam A (10% solution)200 μL0.002%TRIS (1M)10 mL100 mMEDTA (0.5M)20 μL0.1 mMHydrochloric acid (HCl)q.s. to adjust pH to 6.9—Nuclease-free waterq.s. to 100 mL—Magnetic NACM beads80 mg0.8 mg / mL —

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PUM

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Abstract

The invention is directed to compositions and methods for rapidly and efficiently extracting nucleic acids and / or targeted nucleic acids sequences from biological samples. The methods of the invention comprise combining the sample with a buffer and magnetic silicon beads and concentrating the beads with a magnet or other electrical field. Liquid may be removed, or not, and an alkaline buffer is added followed by magnetic carboxy beads in a binding buffer so that nucleic acids transfer to the carboxy beads, which can be easily and quickly isolated once again with a magnet. Total nucleic acid extraction is greatly enhanced. Extracted nucleic acids can be analyzed, for example, by PCR wherein the nucleic acids can be identified and characterized. Carboxy beads may also contain a ligand so as to target specific nucleic acid sequences. The invention is also directed to kits comprising the tools and compositions for performing the methods of the invention.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application No. 62 / 232,666 filed Sep. 25, 2015, and U.S. Provisional Application No. 62 / 161,527 filed May 14, 2015, the entirety of each of which is specifically incorporated herein.FIELD OF THE INVENTION[0002]The invention is directed to rapid methods for the extraction of total and / or targeted nucleic acids from a biological sample and, in particular, to tools, methods and compositions containing components that facilitate concentration and isolation / extraction of nucleic acids from samples.BACKGROUND OF THE INVENTION[0003]The ability to maintain the integrity of nucleic acids in a biological sample (and in particular, those contained in diagnostic samples obtained from human patients), whether the specimen is taken in a remote field location, a doctor's office or in a laboratory, often determines whether the nucleic acids can be successfully analyzed. Typically, nucleic acids in a biologic...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/686C12N15/1013
Inventor FISCHER, GERALD W.DAUM, LUKE T.
Owner LONGHORN VACCINES & DIAGNOSTICS LLC
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